Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Inactivated whole-virion (WV) vaccine retains the protein composition and the structure of the original virus. (A) Scheme of inactivated WV (Gamma strain) production. (B) VeroE6/TMPRSS2 cells were treated with medium as a negative control, inactivated WV (undiluted) and live virus (diluted 10 4 -fold with medium) at 37 °C for 3 days, followed by plaque counting. (C, D) Inactivated WV were validated using (C) SDS-PAGE; left: marker, right: sample and (D) western blotting; left: marker, right: S protein and M protein. Each experiment was performed in duplicate. (E) TEM micrograph of SARS-CoV-2 virus particles, Scale bar, 100 nm.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques: Virus, Negative Control, SDS Page, Marker, Western Blot

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Nasal administration of the inactivated whole-virion (WV) vaccine induces S protein-specific IgG in serum and IgA in nasal wash. (A–H) Mice were immunized twice with inactivated WV intranasally (i.n.) or with inactivated WV and alum subcutaneously (s.c.). Unimmunized mice were used as controls (cont.). (A–F) Endpoint titers of S protein-specific IgG in (A) serum, (B) bronchoalveolar lavage fluid (BALF), and (C) nasal wash, and endpoint titers of S protein-specific IgA in (D) serum, (E) BALF, and (F) nasal wash were evaluated. (G, H) Measurement of neutralization against vesicular stomatitis virus-based pseudotyped viruses displaying Gamma spike of SARS-CoV-2 in (G) serum samples and (H) nasal wash samples. The significance of differences in the (G) 50-fold-diluted serum samples and (H) 4-fold-diluted nasal wash samples was evaluated. The data is presented as mean ± SD. Each experiment was performed in duplicate. (A–H) n = 5 per group. (A–H) * P < 0.05; ** P < 0.01 as indicated by Tukey’s test. ns, not statistically significant.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques: Neutralization, Virus

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Nasal administration of the inactivated whole-virion (WV) vaccine protects against upper and lower respiratory tract infections caused by SARS-CoV-2. (A–E) Mice were immunized twice with inactivated whole-virion (WV) vaccine intranasally (i.n.) or with inactivated WV and alum subcutaneously (s.c.). Unimmunized mice were used as controls (cont.). (A) Mice were intranasally infected with SARS-CoV-2 MA10 (5 × 10 4 PFU) in 5 μL of PBS and the SARS-CoV-2 titer in nasal turbinate was assayed using the plaque assay. (B–E) Mice were intranasally infected with SARS-CoV-2 MA10 ( B, C ; 5 × 10 4 PFU/mouse, D, E ; 2 × 10 5 PFU/mouse) in 20 μL of PBS; (B, D) body weight loss and (C, E) survival were monitored. The data is presented as the mean ± SD. Each experiment was performed in duplicate. (A) n = 5 per group. (B, C) n = 4–5 per group. (D, E) n = 10 per group. (A, B, D) * P < 0.05; ** P < 0.01 as indicated by Tukey’s test. (C, E) ** P < 0.01 vs. the control group as indicated by comparing Kaplan–Meier curves using the Log rank test.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques: Infection, Plaque Assay

Journal: Frontiers in Immunology

Article Title: A nasal vaccine with inactivated whole-virion elicits protective mucosal immunity against SARS-CoV-2 in mice

doi: 10.3389/fimmu.2023.1224634

Figure Lengend Snippet: Systemic priming by mRNA vaccine followed by intranasal boosting with inactivated whole-virion (WV) vaccine induces S protein-specific IgG in serum and IgA in nasal wash. (A–C) Mice were immunized intranasally (i.n.) with inactivated WV or intramuscularly (i.m.) with 1 μg of mRNA vaccine encoding SARS-CoV-2 Spike. (A) Graphical illustration of the experimental procedure. (B, C) Endpoint titers of S protein-specific (B) IgA in the nasal wash and (C) IgG in the serum were evaluated. The data is presented as the mean ± SD. Each experiment was performed in duplicate. (B, C) n = 7–11 per group. (B, C) ** P < 0.01 as indicated by Tukey’s test. ns, not statistically significant.

Article Snippet: The transferred membrane was incubated with rabbit anti-SARS-CoV-2 (2019-nCoV) spike antibody (Polyclonal IgG; Sino Biological).

Techniques:

Journal: RNA Biology

Article Title: Role of small intronic RNAs in the crosstalk between immune cells and β-cells during type 1 diabetes development

doi: 10.1080/15476286.2026.2645442

Figure Lengend Snippet: Identification of small RNAs transferred in vivo from CD4 + /CD25 − T cells to pancreatic β-cells. (A) schematic representation of in vivo T cell adoptive transfer and identification of EU-tagged RNA in pancreatic β-cells. As previously described , CD4 + /CD25 − T cells were purified from NOD.Cg-tg (TcraBDC2.5, TcrbBDC2.5) mice and were incubated with 200 μM ethynyl uridine (EU) for 48 h. The labelled T cells were injected in the tail vein of NOD.CB-17-Prkdc.Scid/Rj mice. Saline solution was injected intravenously as control. The pancreatic islets were isolated after 48 h and β-cells purified by FACS. EU-tagged RNAs were biotinylated using a Click-iT nascent RNA capture kit and then isolated on streptavidin-coated beads. The eluted RNA was used for library preparation and small RNA sequencing. (B) plot showing differential expression of small RNA fragments (16–29 nucleotides) in EU-tagged RNA extracted from the receiving β-cells. Each point represents a small RNA fragment, with the x-axis displaying Fold changes and the y-axis -log 10 adjusted p -values. Blue points highlight the three upregulated sequences of sinR-D and the redpoint sinR-T. As internal control, C. elegans miR-238 RNA sequence was spiked into each sample, and its EU-tagged sequence was used for normalization across samples. (C) to demonstrate widespread expression of the selected sinRNAs, tissue samples were collected from C57BL6 mice and sinRNA levels were measured using qRT-PCR. The relative abundance of sinRnas in each tissue was normalised to Let7a. The results are expressed as relative fluorescent units (RFU).

Article Snippet: Further, 50 μL of streptavidin dynabeads ( M -280 Streptavidin, 11206D, Invitrogen) was added to the samples and incubated again for 1 h at room temperature on a vertical rotator.

Techniques: In Vivo, Adoptive Transfer Assay, Purification, Incubation, Injection, Saline, Control, Isolation, RNA Sequencing, Quantitative Proteomics, Sequencing, Expressing, Quantitative RT-PCR

Journal: RNA Biology

Article Title: Role of small intronic RNAs in the crosstalk between immune cells and β-cells during type 1 diabetes development

doi: 10.1080/15476286.2026.2645442

Figure Lengend Snippet: Mode of action of sinRNAs. (A) Western blot analysis demonstrating the enrichment of Ago2 protein in MIN6 cells incubated with either biotinylated sinRNA mimics or the corresponding scrambled control sequences, followed by pull-down with streptavidin beads. Input lane represents 10% of the total cell lysate. Data shown is a representative image from three independent experimental replicates. (B) MIN6 cells were transfected with a plasmid encoding Flag-tagged Ago2. The small non-coding RNAs immunoprecipitated using an anti-Flag antibody were analyzed by qRT-PCR. (C) volcano plot representing differentially expressed proteins identified by proteomic analysis of pancreatic islets overexpressing both sinR-D2 and sinR-D3 compared to control cells transfected with length-matched scrambled sequences. Significantly upregulated proteins are shown in red, and significantly downregulated proteins are shown in blue ( p < 0.05).

Article Snippet: Further, 50 μL of streptavidin dynabeads ( M -280 Streptavidin, 11206D, Invitrogen) was added to the samples and incubated again for 1 h at room temperature on a vertical rotator.

Techniques: Western Blot, Incubation, Control, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Kefir peptides mitigate bleomycin-induced pulmonary fibrosis in mice through modulating oxidative stress, inflammation and gut microbiota.

doi: 10.1016/j.biopha.2024.116431

Figure Lengend Snippet: Fig. 5. Pretreatment KPs attenuates the expression of fibrotic-related genes in the bleomycin-induced pulmonary fibrosis mouse model. Mice pre-treated with water or KPs for 4 days following intratracheal injection of bleomycin for 21 days. Lung tissues were harvested to detect fibrotic-related mRNA, including (A) Fn1, (B) Col3a1, (C) Col1a1, (D) Timp1, and (E) Ctgf expression by quantitative RT-PCR (n=5 mice per group). Values are normalized to the β-actin gene and are expressed relative to the control group. (F) Western blot analysis was performed to analyze the expression of Collagen I and fibronectin in the lung tissues from each group (n=4–5 mice per group). *p < 0.05, **p < 0.01, and ***p < 0.001. All the statistical information and details of tests for each figure are available in the supple mentary files.

Article Snippet: After transferring to PVDF membranes, the membranes were blocked with 5% nonfat dry milk in TBS-T (0.1% Tween-20 in TBS buffer) for 1 h. Proteins were then probed overnight at 4 ◦C with the following antibodies at appropriate dilutions: 1:2000 dilution of Fibronectin and Collagen type I (Proteintech) and 1:25,000 dilution of β-actin (Novus Biologicals, Centennial, CO, USA).

Techniques: Expressing, Injection, Quantitative RT-PCR, Control, Western Blot

Journal: Biomaterials

Article Title: Dual inhibition of oxidative phosphorylation and glycolysis using a hyaluronic acid nanoparticle NOX inhibitor enhanced response to radiotherapy in colorectal cancer.

doi: 10.1016/j.biomaterials.2025.123437

Figure Lengend Snippet: Fig. 3. Combination of HANP/GKT831 inhibited tumor progression and impacted metabolism with radiotherapy. HANP/GKT831 enhanced sensitivity to radiation and suppressed cell migration and invasion in the MC38 mouse tumor cells. A. Colony formation assay detected cell colonies resistant to 2 Gy of RT (n = 3). B. Transwell migration assay evaluated cell migration following the treatments. Cells were treated with 0.01 μM of conventional GKT831 or HANP/GKT831 con taining 0.01 μM of equivalent doses of GKT831. Quantification of 2D migration assay after 48 h in culture (n = 5). HANP/GKT831 reduced the protein levels of representative signal molecules in glycolysis, mitochondrial OXPHOS, and DNA repair pathways. C. The levels of Western blot analysis of NOX1, NOX4, glycolysis related (Hexokinase 2 and PKM2), and mitochondrial OXPHOS associated proteins (MT-ATP6 and MT-ND1). D. The protein levels in the cell cycle and DNA repair pathway, Cyclin D1, MSH6, and CHK1 proteins, were examined from the treated tumor lysates by Western blot analysis. β-actin served as the loading control. Similar results were obtained from at least 3 repeated studies. Student’s t-test: *p < 0.05 and ****p < 0.0001.

Article Snippet: After transferring to a polyvinylidene difluoride and blocking in 5 % of milk in tris-buffered saline (TBS) for 1 h, the blots were probed with rabbit anti-NOX1 antibody (MBS9609001, MYBioSource, San Diego, CA, USA), rabbit anti-NOX4 antibody (MBS820230, MYBioSource), anti-Hexokinase II rabbit monoclonal antibody (C64G5, Cell Signaling Technology), anti PKM2 XP rabbit monoclonal antibody (D78A4, Cell Signaling Technology), rabbit anti-MT-ATP6 antibody (70262, Cell Signaling Technology), rabbit anti-MT-ND1 antibody (NBP2-94462, NOVUS, MO, USA), anti-Cyclin D1 rabbit monoclonal antibody (2978, Cell Signaling Technology), rabbit anti-MSH6 antibody (MBS9626584, MYBioSource), rabbit anti-Phospho-Chk1 (Ser345) antibody (#2341, Cell Signaling Technology), hamster anti MCP-1 (CCL2) antibody (505911, Biolegend, San Diego, CA, USA), rabbit antiCXCL10 monoclonal antibody (701225, Invitrogen), anti-β-Actin mouse monoclonal antibody (A5316, Sigma-Aldrich).

Techniques: Migration, Colony Assay, Transwell Migration Assay, Western Blot, Control

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2 and TET3 associate with the O -GlcNAc transferase OGT and promote GlcNAcylation. ( A ) Silver stain gel of HaloTag-TET protein complex isolations and HaloTag alone control (Ctrl). Protein pulldowns were performed from HEK293T cells overexpressing the indicated HT constructs (see Materials and methods and for details). As not all of the indicated complex isolations were performed at the same time, two separate silver stain gels were run, as shown in this panel. ( B ) Table of transcriptional or chromatin protein interactors found in the various HaloTag-TET isolations. Spectral counts for each interacting protein are shown for biological replicates. TET1, but not TET2, as previously reported ( ; ), shows interaction with SIN3A. OGT interacts with all TET proteins, though it is most highly abundant with TET2 and TET3. ( C ) Detection of OGT by western blotting from HT-TET2 and HT-TET3 pulldowns from ( A ). The indicated pulldowns were probed with an anti-OGT antibody to detect the presence of OGT. OGT and beta-Actin shown as input loading controls. ( D ) TET2 and TET3 co-immunoprecipitate (CoIP) with endogenous OGT from untransfected HEK293T cells. Cell extracts were immunoprecipitated with anti-OGT or rabbit IgG and probed with antibodies against the indicated proteins. An IP control of OGT alone is shown to demonstrate specific capture and enrichment of OGT. Inputs loading controls are shown for all. Note that in this experiment very weak expression of TET2 relative to TET3 is observed. ( E ) The global level of hmC does not change after cell treatment with Alloxan or PUGNAc. Dot blot quantification of global hmC after the indicated treatments. The hmC content is normalized with respect to the input DNA and to mock-treated cells, where the ratio is set at 1.00. Error bars indicate s.d. of three independent experiments. As controls, western blots using anti- O -GlcNAc antibody show the expected decrease in GlcNAcylation with Alloxan and increase with PUGNAc. HDAC1 input loading controls are also shown. Vertical line indicates juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( F ) Global decrease in GlcNAcylation is observed in TET2/3 knockdowns. Left: TET2 kd or TET3 kd show decreased GlcNAc activity. Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl, RNAi TET2, or RNAi TET3, and UDP-[ 3 H]GlcNAc incorporation was measured. The amount incorporated into the control cells was set at 1. Error bars indicate s.d. of three independent experiments (* P <0.05). Right: Nuclear extracts were prepared from HEK293T cells expressing RNAi Ctrl or RNAi TET2/3 and global GlcNAcylation was visualized with an antibody against O -GlcNAc. HDAC1 input loading control is also shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Silver Staining, Control, Construct, Western Blot, Immunoprecipitation, Expressing, Dot Blot, Activity Assay

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3–OGT show genomic co-localization around TSSs and impact on H3K4me3 and transcriptional activation. ( A ) Left: Venn diagrams indicating significant overlap of TET2 and OGT bound regions (left part; P -value<10 −10 ) identified after HaloCHIP-Seq in HEK293T cells expressing HT-TET2, or HT-OGT. Right: TET2–OGT targets are primarily found at TSSs and CpG-rich sequences. Similar profiles were also observed for TET3–OGT . ( B ) An analysed subset of TET2–TET3–OGT targets show a lack of DNA methylation and hydroxymethylation, yet display GlcNAcylation. qPCR analysis of TET2–TET3–OGT binding and non-binding regions after MeDIP (top), hMeDIP (middle), or ChIP with an anti- O -GlcNAc antibody (bottom). ‘% Input' represents real-time qPCR values normalized with respect to the input chromatin. Known methylated and hydroxymethylated regions are shown as positive controls in MeDIP and hMeDIP panels.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Binding Assay, Methylated DNA Immunoprecipitation, Methylation

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: ( C ) TET2/3–OGT targets in HEK293T cells are enriched for H3K4me3 as depicted in a Venn diagram; P -value<10 −10 . ( D ) Examples of HaloCHIP-Seq OGT, TET2, TET3, and ChIP-Seq H3K4me3 profiles (UCSC tracks). ( E ) Decreased levels of H3K4me3 in TET2 kd cells. Upper-left: decrease in the normalized number of H3K4me3 reads in TET2/3–OGT-binding regions in TET2 kd cells versus control RNAi-treated cells. Upper-right: pie chart showing the percentage of TET2–TET3–OGT binding regions with a statistically significant reduction of the normalized number of H3K4me3 reads for TET2 kd versus control RNAi-treated cells. Lower-part: examples of H3K4me3 ChIP-Seq profiles (UCSC tracks) in TET2–TET3–OGT-binding regions for the RNAi control versus TET2 kd sample. ( F ) Western blot showing global decrease in H3K4me3 in a TET2/3 double kd cells. Lysates from mock HEK293T RNAi kd or TET2/3 kd cells were probed for H3K4me3 using an anti-H3K4 antibody in western blot. Tubulin is shown as a loading control. ( G ) OGT activity is important for H3K4me3. Cell extracts were prepared from HEK293T cells treated with or without the OGT inhibitor Alloxan, and then western blots for H3K4me3 were performed. HDAC1 and H3 are shown as loading controls and a western blot against O -GlcNAc was used to monitor specific GlcNAcylation inhibition by Alloxan. Vertical lines indicate juxtaposition of lanes non-adjacent within the same blot, exposed for the same time. ( H ) Decreases in transcription are observed in both TET2/3 knockdowns and an OGT knockdown. The indicated target genes (which showed decrease in H3K4me3 after TET2 kd; cf. E ) and negative controls (unbound TET2/3–OGT–H3K4me3 targets), were analysed by RT–qPCR in HEK293T cells subjected to the various listed RNAi treatments. Independent experiments were performed in duplicates.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: ChIP-sequencing, Binding Assay, Control, Western Blot, Activity Assay, Inhibition, Knockdown, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: TET2/3 promotes GlcNAcylation of HCF1, and both TET and OGT activity favour the integrity of SET1/COMPASS and SETD1A binding to chromatin. ( A ) Mass spectrometry reveals HCF1, a known target of OGT and component of SET1/COMPASS , as an interacting partner of HT-TET2 and HT-TET3. Biological duplicates and respective spectral counts (SpC) for HCF1 are shown. ( B ) Protein pulldowns of HT-OGT coupled with mass spectrometry identify HCF1, TET2, TET3, and all components of SET1/COMPASS as partners of OGT. Biological duplicates and SpC for each protein identified are shown for HT-OGT and Ctrl isolations. ( C ) The interaction of HCF1 and SET1/COMPASS components with HT-OGT depends on O -GlcNAc activity. Plot showing average SpCs for HCF1 and SET1/COMPASS components isolated from HT-OGT pulldowns of untreated (grey bars) and Alloxan-treated (green bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( D ) The interaction of HT-SETD1A with SET1/COMPASS components and OGT is reduced by a TET2/3 double kd. Plot showing average SpCs for SET1/COMPASS components and OGT isolated from HT-SETD1A pulldowns of control RNAi-treated (grey bars) and TET2/3 kd (blue bars) HEK293T cells. Error bars represent s.d. of biological duplicates. Representative NSAF plots are shown in . ( E ) A significant reduction in HCF1 GlcNAcylation is observed after TET2/3 double kd. Upper diagram shows a schematic representation of full-length HCF1 and its domains . The GlcNAcylated peptides identified by mass spectrometry from HT-SETD1A isolations from control RNAi-treated and TET2/3 kd cells are indicated below. The full-length HCF1 amino-acid sequence (NP_005325.2) shows the corresponding GlcNAcylated peptides highlighted in yellow with RNAi Ctrl on the left and RNAi TET2/3 kd on the right. ( F ) Bioluminescence resonance energy transfer (BRET) assays show reduction of SETD1A binding to histone H3.3 in the presence of an OGT inhibitor and in TET2/3 kd cells. Upper diagram showing the schematic of BRET energy transfer upon binding of a NanoLuc-SETD1A fusion donor and fluorescently labelled Histone H3.3-HaloTag fusion acceptor in live HEK293T cells (see Materials and methods for experimental details and calculation of BRET). Left: BRET measurements were calculated without treatment (grey) or with Alloxan treatment (green). Right: BRET measurement for RNAi control (grey) or RNAi TET2/3 (blue). Biological triplicates ±s.d. are shown.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Mass Spectrometry, Isolation, Control, Sequencing, Bioluminescence Resonance Energy Transfer

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Tet2 knockout mouse tissue shows that Tet2 is needed for global GlcNAcylation and H3K4me3 at target promoters. ( A ) Genome-wide co-localization of endogenous Tet2 with O -GlcNAc and H3K4me3 at promoters and CpG-rich regions. Venn diagrams are shown ( P -value overlap<10 −10 ) as well as the indicated genome-wide distribution. ( B ) Tet2, O -GlcNAc, and H3K4me3 are enriched at many active genes, mirroring the presence of RNA Pol II. Upper panel: Venn diagram showing the overlap of Tet2 and O -GlcNAc with RNA Pol II ( P -value overlap <10 −10 ); lower panel: Box plots showing the reads density at targets and non-targets (others) for ChIP-Seq RNA Pol II or RNA-Seq in mouse bone marrow. ( C ) Global decrease in GlcNAcylation is observed in Tet2 knockout mouse bone marrow. Mouse bone marrow tissues with or without a Tet2 knockout were analysed by western blot for O -GlcNAc levels using an anti- O -GlcNAc antibody. HDAC1 is shown as loading control. ( D ) ChIP-Seq for H3K4me3 in Tet2 knockout mouse tissues shows reduced global H3K4me3 at target promoters. Overall impact on H3K4me3 peak significance (−log( P -value) of the peaks) between wild-type and Tet2 knockout bone marrow is shown. ( E ) Table showing key haematopoietic genes with specifically reduced H3K4me3 in Tet2 knockout as compared to wild type. The location at CpG islands and the promoter class for each is listed. Lower part: example of H3K4me3 ChIP-Seq profiles (UCSC tracks) in wild type versus Tet2 knockout.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Knock-Out, Genome Wide, ChIP-sequencing, RNA Sequencing, Western Blot, Control

Journal: The EMBO Journal

Article Title: TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

doi: 10.1038/emboj.2012.357

Figure Lengend Snippet: Model connecting DNA modifying enzymes, TETs, a master cellular sensor protein, OGT, and a histone modifying complex, SET1/COMPASS. Based on our findings, a hierarchical model of the involved proteins, with the cascade of their respective activities, can be envisaged as followed: (1) The first sequence of events in the cascade is the formation of TET2/3–OGT interaction, which promotes OGT GlcNAcylation on numerous proteins, including HCF1; (2) In a TET-dependent manner, a GlcNAcylated HCF1 is important for the formation of the SET1/COMPASS; (3) In the last step, both TET proteins and OGT activity favour binding of SETD1A to chromatin, an event necessary for histone H3K4me3 and subsequent transcriptional activation.

Article Snippet: 2 μg of mouse monoclonal antibody for H3K4me3 (ab1012; Abcam), 6 μg of mouse monoclonal antibody for O -linked N-acetylglucosamine (ab2739; Abcam), 3 μg of rabbit polyclonal antibody for Tet2 (sc-136926; Santa Cruz), 5 μg of rabbit polyclonal for HCF1 (A301-399A-1; Bethyl Lab), or the respective amount of control antibody was incubated with chromatin overnight at 4°C.

Techniques: Sequencing, Activity Assay, Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of the MNV infection experiment with male BALB/c. Ncf1 90H mice after transferring from the SPF facility to the MNV-infected facility (day 1). b The representative appearance of arthritis in the ankle of hind paws on day 49 and mean arthritis scores from days 1 to 56 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. c The levels of proteinuria ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. d The level of anti-dsDNA, anti-Sm/RNP, anti-phospholipid (anti-PLs) specific antibodies, and β2-GP1 on day 56 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e Correlation with anti-MNV antibodies (OD): maximum arthritis score ( p = 0.0138), proteinuria ( p = 0.0049), anti-dsDNA antibodies ( p = 0.0055) in Ncf1 90H mice on day 56 post of MNV infection ( n = 8 per group). Data were analyzed using the Pearson correlation test. f Enlarged spleen and spleen index in mice ( n = 7/group). The spleen index is defined by the spleen weight (mg) divided by the body weight ( g ) and then multiplied by 10 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. g Representative H&E stained joint and kidney sections (magnification x 10). Histological scoring of joint and kidney inflammation using a scale of 0-3 ( n = 8 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. h Immunofluorescence images (magnification x 20) and histology scores of deposits of IgG and C3 in the glomerulus from wild-type (WT) R90 and 90H mice on day 56 ( n = 4 per group). Data were analyzed using one-way ANOVA and presented as mean ± SEM. i Relative expression of Ifnα and anti-viral ISGs ( Irf1 , Irf7 , Stat1 , Mx1 , Ip10 and Isg15 ) within spleens ( n = 4 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. j Relative expression of Ifnα and anti-viral ISGs ( Irf1 , Irf7 , Stat1 , Mx1 , Ip10 and Isg15 ) within kidneys. The expression of mRNAs was normalized to the housekeeping gene β-actin ( n = 4 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. k Immunoblot analysis of p-JAK1/JAK1 and p-STAT1/STAT1 proteins in the kidneys on day 56 after MNV infection ( n = 6 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Transferring, Staining, Immunofluorescence, Expressing, MANN-WHITNEY, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of MNV infection by gavage. b The quantification of MNV in the small intestine, colon, and spleen on day 7 by RT-qPCR ( n = 9 per group). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. c Titer of anti-MNV antibodies on days 0, 14, 28, 56, 70, and 84 in females (MNV-R90: n = 6; MNV-90H: n = 13) and males (MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. d IgG production in femals (R90: n = 6; 90H: n = 13; MNV-R90: n = 6; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 6; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e The correlation between IgG and anti-MNV antibodies in wild-type Ncf1 R90 mice ( n = 11) and Ncf1 90H mice ( n = 12). Data were analyzed using the Pearson correlation test. f Anti-ssRNA antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13). Data were analyzed using one-way ANOVA and presented as mean ± SEM. g Anti-dsDNA antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. h Anti-Sm/RNP antibodies on day 56 in females (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 5; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. i Proteinuria on day 56 in females (R90: n = 6; 90H: n = 13; MNV-R90: n = 6; MNV-90H: n = 13) and males (R90: n = 5; 90H: n = 6; MNV-R90: n = 5; MNV-90H: n = 6). Data were analyzed using one-way ANOVA and presented as mean ± SEM. j The ratio of Tcm or Tvm cells in CD4 + Foxp3 + in the PPs (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. k, l The ratio of Tfh cells and Tfr cells in CD4 + FOXP3 + cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. m The ratio of Trm cells in CD4 + CD44 hi cells (MNV-R90: n = 11; MNV-90H: n = 18). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. n The ratio of GC-B cells in IgD lo B cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. o The ratio of LLPCs in IgD lo CD138 + Sca-1 + cells (MNV-R90: n = 11; MNV-90H: n = 19). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Quantitative RT-PCR, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: The systemic lupus erythematosus-associated NCF1 90H allele synergizes with viral infection to cause mouse lupus but also limits virus spread

doi: 10.1038/s41467-025-56857-z

Figure Lengend Snippet: a Timeline of MNV infection in male mice by i.v. and i.p. injection. b Anti-MNV antibodies on days 0, 14, 28, and 56. c Anti-ssRNA antibodies on day 21 (R90: n = 6; 90H: n = 6; MNV-R90: n = 11; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM. d IgG production on day 21 (R90: n = 8; 90H: n = 12; MNV-R90: n = 11; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM. e Representative paws on day 21. f Mean arthritis score (MNV-R90: n = 8; MNV-90H: n = 11). Data were analyzed by the Mann-Whitney test (two-tailed) and presented as mean ± SEM. g Arthritis incidence ( n = 9 per group). h Representative HE staining of the joints ( n = 4, magnification x 2.5 and x 10). i Anti-collagen II (COL2) IgG antibodies (R90: n = 7; 90H: n = 7; MNV-R90: n = 11; MNV-90H: n = 20). Data were analyzed using one-way ANOVA and presented as mean ± SEM. j Anti-COL2 IgG2b antibodies (R90: n = 7; 90H: n = 7; MNV-R90: n = 9; MNV-90H: n = 20). Data were analyzed using one-way ANOVA and presented as mean ± SEM. k Anti-COL2 IgM antibodies (R90: n = 8; 90H: n = 11; MNV-R90: n = 11; MNV-90H: n = 18). Data were analyzed using one-way ANOVA and presented as mean ± SEM. The level of anti-COL2 antibodies was measured in mouse sera on day 21 by ELISA. l , m Representative B cell-ELISpot and statistics of total anti-COL2 IgG and IgM ASCs in the spleen were shown (R90: n = 5; 90H: n = 5; MNV-R90: n = 8; MNV-90H: n = 12). Data were analyzed using one-way ANOVA and presented as mean ± SEM.

Article Snippet: After washing, biotinylated goat anti-mouse IgG (1:1000 dilutions, Southern Biotech, 1030-08) or IgG2b (1:1000 dilutions, Southern Biotech, 1090-08) was added to the wells and incubated at RT for 2 h. ExtrAvidin conjugated alkaline phosphatase (Sigma-Aldrich, E2636) diluted in PBS was added and incubated for 30–45 min. 100 μL of substrate BCIP/Nitroblue Tetrazolium (Sigma-Aldrich, B3804) per well (1 tablet in 10 mL ddH 2 O and pre-filtered with 0.45 μm) was added and incubated in the dark for 10–15 min.

Techniques: Infection, Injection, MANN-WHITNEY, Two Tailed Test, Staining, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Resolvin D1 combined with exercise rehabilitation alleviates neurological injury in mice with intracranial hemorrhage via the BDNF/TrkB/PI3K/AKT pathway

doi: 10.1038/s41598-024-83019-w

Figure Lengend Snippet: RvD1 combined with exercise rehabilitation training can activate BDNF/TrκB/p-Akt/PI3K related pathway and increase its protein and mRNA expression. ( a ) Western blot analysis was used to detect the expression levels of BDNF, TrκB, PI3K proteins, and both phosphorylated and total Akt. ( b - d ) Quantitative analysis of BDNF, TrκB, and PI3K protein expressions in brain tissue adjacent to the injection site on the ipsilateral side after the experiment. ( e ) Semiquantitative measurements of the ratio of phosphorylated to total Akt. ( f - i ) Expression levels of BDNF ( f ), TrκB ( g ), PI3K ( h ), and Akt ( i ) mRNAs at the injection site were determined. Western blot and RT-qPCR analyses revealed that the expression levels of BDNF, TrκB, p-Akt, and PI3K in the ICH + RvD1 + exercise training group were significantly higher compared to those in the ICH group. (*Significant vs. ICH group, p < 0.05).

Article Snippet: Equal amounts of extracted proteins was added to 5X loading buffer, heated to 95 ° C for denaturation, and the proteins were separated by electrophoresis in 10% polyacrylamide gel.After transferring the proteins to PVDF membrane by wet transfer method, they were blocked in 5% bovine serum protein for 1 h, then the following primary antibodies were added and incubated overnight at 4 ° C: BDNF (1:4000, 25699-1-AP, Proteintech), AKT (1:4000, 10176-2-AP, Proteintech), phospho-AKT (1:2000, 66444-1-Ig, Proteintech), PI3K (1:1000, 20584-1-AP, Proteintech), TrkB (1:4000, 13129-1-AP, Proteintech), GAPDH (1:4000, GB12002-100, Servicebio).The following day, the primary antibodies were removed, and the PVDF membranes were washed three times with TBST, each wash lasting 5 min.

Techniques: Expressing, Western Blot, Injection, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Resolvin D1 combined with exercise rehabilitation alleviates neurological injury in mice with intracranial hemorrhage via the BDNF/TrkB/PI3K/AKT pathway

doi: 10.1038/s41598-024-83019-w

Figure Lengend Snippet: RvD1 combined with exercise rehabilitation training activates BDNF/TrkB/PI3K/AKT signaling pathway to reduce neuroinflammation after intracerebral hemorrhage and improve neurological prognosis.

Article Snippet: Equal amounts of extracted proteins was added to 5X loading buffer, heated to 95 ° C for denaturation, and the proteins were separated by electrophoresis in 10% polyacrylamide gel.After transferring the proteins to PVDF membrane by wet transfer method, they were blocked in 5% bovine serum protein for 1 h, then the following primary antibodies were added and incubated overnight at 4 ° C: BDNF (1:4000, 25699-1-AP, Proteintech), AKT (1:4000, 10176-2-AP, Proteintech), phospho-AKT (1:2000, 66444-1-Ig, Proteintech), PI3K (1:1000, 20584-1-AP, Proteintech), TrkB (1:4000, 13129-1-AP, Proteintech), GAPDH (1:4000, GB12002-100, Servicebio).The following day, the primary antibodies were removed, and the PVDF membranes were washed three times with TBST, each wash lasting 5 min.

Techniques:

Journal: Scientific Reports

Article Title: Resolvin D1 combined with exercise rehabilitation alleviates neurological injury in mice with intracranial hemorrhage via the BDNF/TrkB/PI3K/AKT pathway

doi: 10.1038/s41598-024-83019-w

Figure Lengend Snippet: The primer sequences for BDNF, TrkB, PI3K, AKT, IL-1β, IL-10, TNF-α, CD206 and GAPDH.

Article Snippet: Equal amounts of extracted proteins was added to 5X loading buffer, heated to 95 ° C for denaturation, and the proteins were separated by electrophoresis in 10% polyacrylamide gel.After transferring the proteins to PVDF membrane by wet transfer method, they were blocked in 5% bovine serum protein for 1 h, then the following primary antibodies were added and incubated overnight at 4 ° C: BDNF (1:4000, 25699-1-AP, Proteintech), AKT (1:4000, 10176-2-AP, Proteintech), phospho-AKT (1:2000, 66444-1-Ig, Proteintech), PI3K (1:1000, 20584-1-AP, Proteintech), TrkB (1:4000, 13129-1-AP, Proteintech), GAPDH (1:4000, GB12002-100, Servicebio).The following day, the primary antibodies were removed, and the PVDF membranes were washed three times with TBST, each wash lasting 5 min.

Techniques: Sequencing

Journal: Physiological Reports

Article Title: Evidence for the involvement of NADPH oxidase in adenosine receptor-mediated control of coronary flow using A 1 and A 3 knockout mice

doi: 10.1002/phy2.70

Figure Lengend Snippet: (A) Representative blots for changes in protein expression for Nox1, 2, and 4 induced by adenosine in WT hearts. (B) Representative blots and densitometric analysis for changes in phosphorylation of p47-phox induced by adenosine in WT hearts. Data are expressed as mean ± SEM ( n = 4). * P < 0.05, significantly different compared to control using unpaired t -test.

Article Snippet: Total and phospho proteins were separated on the same gel, transferred, and probed with anti-p47-phox rabbit polyclonal IgG (Santa Cruz Biotechnology) as previously mentioned in immunoblotting.

Techniques: Expressing, Phospho-proteomics, Control

Journal: The Journal of investigative dermatology

Article Title: Human mitochondrial NAD(P)(+)-dependent malic enzyme participates in cutaneous melanoma progression and invasion.

doi: 10.1038/jid.2014.385

Figure Lengend Snippet: Figure 4. AMP-activated protein kinase (AMPK) activation as the mechanism for melanoma cell growth inhibition in response to ME2 depletion. (a) Silencing ME2 diminished ATP production in melanoma cells. ATP levels in A2058 cells were measured and normalized to the respective protein concentration. (b) Increased reactive oxygen species levels in A2058 ME2-knockdown cells as determined by flow cytometry using 2’,7’- dichlorodihydrofluorescein diacetate (DCF). Each histogram is representative of three independent experiments. (c) Western blot analysis was used to determine the protein levels of p-AMPK (Thr172), total AMPK, acetyl Co-A carboxylase (ACC), p53, and p21. Phospho Ser-79 ACC is a biomarker of AMPK activity. (d) Western blot of p-AMPK, total AMPK, ACC, p53, and p21 in indicated melanoma cell lines transfected with control vector or with ME2- expressing vector. The level of ME2 protein after normalization with a-actinin is presented as fold change.

Article Snippet: After transferring the proteins to a polyvinylidine difluoride membrane (Millipore, Bedford, MA), the proteins were detected using antibodies against alpha-actinin (ACTN), p53, p21 (Santa Cruz Biotechnology, Santa Cruz, CA), ME2 (Sigma-Aldrich), and AMPK and acetyl Co-A carboxylase antibody sampler kit (Cell Signaling, Beverly, MA).

Techniques: Activation Assay, Inhibition, Protein Concentration, Knockdown, Cytometry, Western Blot, Biomarker Discovery, Activity Assay, Transfection, Control, Plasmid Preparation, Expressing

Journal: Cell Death & Disease

Article Title: The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

doi: 10.1038/s41419-019-1988-0

Figure Lengend Snippet: a Immunofluorescence in NTERTs transiently transfected with Dsg3 specific or scrambled siRNA for 2d showed significantly increased nuclear p53 relative to control ( n = 7, pooled from 2 independent experiments). Scale bars, 20 µm. b Western blotting for p53 and its targets, p21 WAF1/CIP1 /Bax, in NTERTs with Dsg3 knockdown indicated a moderate but significant increase of p53 ( n = 3–4). c Biochemical fractionation of NTERTs with or without Dsg3 knockdown (RNAi). Increased p53 and p21 WAF1/CIP1 in RNAi samples, especially in the nuclear fraction compared to control. d Western blots for the indicated antibodies in lysates with single (Dsg3) and double (Dsg3/p53) knockdown. e Western blotting analysis of NTERT cell lines with transduction of GIPZ Lentiviral shRNAs, including non-target (NT) and three hits targeting different regions in the Dsg3 gene. Cells with transduction of one (Hit-1), among three hits, exhibited Dsg3 knockdown with concomitant induction of p53/ p21 WAF1/CIP1 without and with either MG-132 (25 µM for 3 h) or UVB irradiation, as compared to NT and negative Hit controls. f Immunofluorescence analysis indicated a marked increase of p53 in both the nucleus and cytoplasm in cells with transduction of Hit-1 compared to NT and other negative Hit controls. g , h Western blotting analysis in NTERTs with Dp or E-cadherin knockdown shows distinct protein expression profiles for p53/p21 WAF1/CIP1 /Bax. Cells treated without and with UVB irradiation were shown here (see Dsg3 KD + UV in Fig. below). (mean ± s.d., * p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The following mouse (m) and rabbit (r) monoclonal/polyclonal antibodies (Abs) were used: Dsg3 mAb against the N-terminus (5H10) (sc-23912, Santa Cruz); Dsg3 rAb against the C- terminus (H145) (sc-20116, Santa Cruz); p53 mAb (DO-1) (ab1101, Abcam); p53 rAb (C-19) (sc-1311-R, Santa Cruz); MDM2 rAb (EP16627) (ab178938, Abcam); phospho MDM2 rAb (S166) (ab131355, Abcam); p21 WAF1/CIP1 rAb (C-19) (sc-397, Santa Cruz); Bax mAb (sc-20067, Cell Signaling); Caspase3 rAb (clone C92-605, RUO) (14C10, BD Biosciences); Caspase3 rAb (9662 S, Cell Signaling); active Caspase3 rAb (ab49822, Abcam); Desmoplakin rAb (sc-33555, Santa Cruz); Plakoglobin mAb (PG51, Progen); Dsc2 rAb (610120, Progen); Dsg2 mAb (33-3D) was kindly received from Prof. David Garrod; E-Cadherin mAb (HECD-1) (ab1416, Abcam); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rAb (14c10, Cell Signaling); HSC70 mAb (B6:sc-7298, Santa Cruz); β-actin mAb (8H10D10, Cell Signaling); anti-53BP1 (05–726, Upstate); anti-ATM (phospho S1981) (ab81292, Abcam); anti-ATM (ab32420, Abcam); anti-CHK2 (phospho T68) (ab32148, Abcam); anti-CHK2 (ab109413, Abcam).

Techniques: Immunofluorescence, Transfection, Control, Western Blot, Knockdown, Fractionation, Transduction, Irradiation, Expressing

Journal: Cell Death & Disease

Article Title: The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

doi: 10.1038/s41419-019-1988-0

Figure Lengend Snippet: a Western blotting of siRNA pre-treated NTERT cells with and without UVB irradiation for the indicated proteins with the quantitation shown on the right ( n = 3 biologically independent samples, * p < 0.05). b Western blotting for p53/p21 WAF1/CIP1 in cells treated with or without actinomycin D (Act D, 5 nM) and mitomycin C (MMC, 5 µg/ml) for 24 h, respectively. The quantitation data are shown on the right. Enhanced expression of p53/p21 WAF1/CIP1 was shown in cells with Dsg3 knockdown and treated with drugs. c Mechanical stretching induced increased expression of p53 and p21 WAF1/CIP1 / Bax in Dsg3 KD cells. The siRNA pre-treated cells were seeded at confluent density in BioFlex plates and then subjected to cyclic strain (TX-5000, 20% amplitude, 1 Hz) for 4 h the following day. Lysates were extracted either immediately after strain or 2 h and 24 h later, respectively, after transferring to a stationary state, along with static control cells. d Western blotting analysis for PCNA and cyclin A in siRNA treated cells with and without UV. e Western blots for the indicated proteins upstream of p53 as well as phospho-p53-S20 in siRNA pre-treated cells with and without UVB (30 mJ/cm 2 ). f Qnatitation of 53BP1 nuclear staining ( n = 10, mean ± S.D., ** p < 0.01, *** p < 0.001). g Western blotting in siRNA transfected cells treated in the presence and absence of ATM inhibitor KU55933 (20 µM) and p38 MAPK inhibitor SB203580 (20 µM), respectively. All cells were exposed to UVB (30 mJ/cm 2 ) in this case. Cells were treated with drugs 1 h before UV and were grown overnight before lysate extraction. h The expression of p53 in the same samples as phosphorylated ATM and CHK2 (the last two corrected for total protein) following the indicated drug treatments in Dsg3-depleted cells. The data are expressed as the band intensities in the Dsg3-depleted cells relative to the corresponding scrambled siRNA controls, which were normalized to 1

Article Snippet: The following mouse (m) and rabbit (r) monoclonal/polyclonal antibodies (Abs) were used: Dsg3 mAb against the N-terminus (5H10) (sc-23912, Santa Cruz); Dsg3 rAb against the C- terminus (H145) (sc-20116, Santa Cruz); p53 mAb (DO-1) (ab1101, Abcam); p53 rAb (C-19) (sc-1311-R, Santa Cruz); MDM2 rAb (EP16627) (ab178938, Abcam); phospho MDM2 rAb (S166) (ab131355, Abcam); p21 WAF1/CIP1 rAb (C-19) (sc-397, Santa Cruz); Bax mAb (sc-20067, Cell Signaling); Caspase3 rAb (clone C92-605, RUO) (14C10, BD Biosciences); Caspase3 rAb (9662 S, Cell Signaling); active Caspase3 rAb (ab49822, Abcam); Desmoplakin rAb (sc-33555, Santa Cruz); Plakoglobin mAb (PG51, Progen); Dsc2 rAb (610120, Progen); Dsg2 mAb (33-3D) was kindly received from Prof. David Garrod; E-Cadherin mAb (HECD-1) (ab1416, Abcam); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rAb (14c10, Cell Signaling); HSC70 mAb (B6:sc-7298, Santa Cruz); β-actin mAb (8H10D10, Cell Signaling); anti-53BP1 (05–726, Upstate); anti-ATM (phospho S1981) (ab81292, Abcam); anti-ATM (ab32420, Abcam); anti-CHK2 (phospho T68) (ab32148, Abcam); anti-CHK2 (ab109413, Abcam).

Techniques: Western Blot, Irradiation, Quantitation Assay, Expressing, Knockdown, Transferring, Control, Staining, Transfection, Extraction

Journal: Cell Death & Disease

Article Title: The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

doi: 10.1038/s41419-019-1988-0

Figure Lengend Snippet: a Western blots of siRNA-transfected cells with and without treatment of MG-132 (25 µM) for 3 h. GAPDH and HSC70 were the loading controls. b Protein turnover analysis for p53, as well as MDM2, indicated reduced p53 turnover accompanied by MDM2 stabilization in Dsg3 depleted cells. The above is the timeline of the experiment. The band density for each blot was normalized against the loading control in each sample and then against the one at 0 min time point in each condition. The calculated half-life for p53 and MDM2 were shown in the graphs. c Flow cytometric analysis of cell viability with live cells (Zombie NIR −ve /Caspase-3 −ve ), Zombie NIR −ve /Caspase-3 +ve , both positive (Zombie NIR +ve /Caspase-3 +ve ) and Zombie NIR +ve /Caspase-3 −ve in NTERTs with and without Dsg3 knockdown, grown at 100% or ~40% confluences (the represented data of 3 independent attempts). d Protein expression in cutaneous keratinocytes T8 (p53 null, with p53 transfection) Vect control and Dsg3 overexpression (D3) that showed suppression of p53/p21 WAF1/CIP1 in D3 cells compared to Vect cells. e RT-qPCR analysis of p53 expression (mean ± s.e.m.) in T8 cell lines ( n = 3 independent assays of duplicate in each test). f p53 luciferase assay (mean ± s.d.) in T8 cell lines ( n = 3, a representative of two independent experiments). The comparison was via unpaired two-sided student t -test. (** p < 0.01, ** p < 0.01, *** p < 0.001)

Article Snippet: The following mouse (m) and rabbit (r) monoclonal/polyclonal antibodies (Abs) were used: Dsg3 mAb against the N-terminus (5H10) (sc-23912, Santa Cruz); Dsg3 rAb against the C- terminus (H145) (sc-20116, Santa Cruz); p53 mAb (DO-1) (ab1101, Abcam); p53 rAb (C-19) (sc-1311-R, Santa Cruz); MDM2 rAb (EP16627) (ab178938, Abcam); phospho MDM2 rAb (S166) (ab131355, Abcam); p21 WAF1/CIP1 rAb (C-19) (sc-397, Santa Cruz); Bax mAb (sc-20067, Cell Signaling); Caspase3 rAb (clone C92-605, RUO) (14C10, BD Biosciences); Caspase3 rAb (9662 S, Cell Signaling); active Caspase3 rAb (ab49822, Abcam); Desmoplakin rAb (sc-33555, Santa Cruz); Plakoglobin mAb (PG51, Progen); Dsc2 rAb (610120, Progen); Dsg2 mAb (33-3D) was kindly received from Prof. David Garrod; E-Cadherin mAb (HECD-1) (ab1416, Abcam); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rAb (14c10, Cell Signaling); HSC70 mAb (B6:sc-7298, Santa Cruz); β-actin mAb (8H10D10, Cell Signaling); anti-53BP1 (05–726, Upstate); anti-ATM (phospho S1981) (ab81292, Abcam); anti-ATM (ab32420, Abcam); anti-CHK2 (phospho T68) (ab32148, Abcam); anti-CHK2 (ab109413, Abcam).

Techniques: Western Blot, Transfection, Control, Knockdown, Expressing, Over Expression, Quantitative RT-PCR, Luciferase, Comparison

Journal: Cell Death & Disease

Article Title: The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

doi: 10.1038/s41419-019-1988-0

Figure Lengend Snippet: a Immunofluorescent staining in the back skin of Dsg3 −/− and Dsg3 +/− (heterozygous littermate) mice showed elevated signals for the indicated proteins in the hair follicles of Dsg3 −/− mice compared to heterozygous littermate, though no positive staining was observed in the epidermis ( n = 2 mice per group, aged 8–12 weeks). Some fibroblasts in the dermis were also shown positive staining of p53. Epi: epidermis, HF: hair follicle. The inset in the top right panel highlight cells with double positive staining for p53 and active caspase-3 in Dsg3 null skin. Scale bar, 20 µm. b Tables summarize the scores of positive hair follicle staining for p53/active caspase-3 and p53/p21 WAF1/CIP1 , respectively. Each hair follicle containing one or more positively stained keratinocytes was scored positive

Article Snippet: The following mouse (m) and rabbit (r) monoclonal/polyclonal antibodies (Abs) were used: Dsg3 mAb against the N-terminus (5H10) (sc-23912, Santa Cruz); Dsg3 rAb against the C- terminus (H145) (sc-20116, Santa Cruz); p53 mAb (DO-1) (ab1101, Abcam); p53 rAb (C-19) (sc-1311-R, Santa Cruz); MDM2 rAb (EP16627) (ab178938, Abcam); phospho MDM2 rAb (S166) (ab131355, Abcam); p21 WAF1/CIP1 rAb (C-19) (sc-397, Santa Cruz); Bax mAb (sc-20067, Cell Signaling); Caspase3 rAb (clone C92-605, RUO) (14C10, BD Biosciences); Caspase3 rAb (9662 S, Cell Signaling); active Caspase3 rAb (ab49822, Abcam); Desmoplakin rAb (sc-33555, Santa Cruz); Plakoglobin mAb (PG51, Progen); Dsc2 rAb (610120, Progen); Dsg2 mAb (33-3D) was kindly received from Prof. David Garrod; E-Cadherin mAb (HECD-1) (ab1416, Abcam); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rAb (14c10, Cell Signaling); HSC70 mAb (B6:sc-7298, Santa Cruz); β-actin mAb (8H10D10, Cell Signaling); anti-53BP1 (05–726, Upstate); anti-ATM (phospho S1981) (ab81292, Abcam); anti-ATM (ab32420, Abcam); anti-CHK2 (phospho T68) (ab32148, Abcam); anti-CHK2 (ab109413, Abcam).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

doi: 10.1038/s41419-019-1988-0

Figure Lengend Snippet: p53 a and cleaved Caspase-3 b immunohistochemistry in oral mucous tissues from PV patients. Significantly enhanced p53 staining was detected in 48% of patients (arrowheads indicate positive nuclear staining whereas arrows indicate predominant cytoplasmic staining), compared to normal controls. Oral mucous cancer was used as positive control here. Asterisks indicate the areas of the blisters. Positive staining of active caspase-3 was also detected in PV patients with the positive p53 staining in oral mucous tissues, especially in the basal and immediate suprabasal layers of stratified squamous epithelium. The positive staining was also detected in cells located in sub-mucous connective tissue (right)

Article Snippet: The following mouse (m) and rabbit (r) monoclonal/polyclonal antibodies (Abs) were used: Dsg3 mAb against the N-terminus (5H10) (sc-23912, Santa Cruz); Dsg3 rAb against the C- terminus (H145) (sc-20116, Santa Cruz); p53 mAb (DO-1) (ab1101, Abcam); p53 rAb (C-19) (sc-1311-R, Santa Cruz); MDM2 rAb (EP16627) (ab178938, Abcam); phospho MDM2 rAb (S166) (ab131355, Abcam); p21 WAF1/CIP1 rAb (C-19) (sc-397, Santa Cruz); Bax mAb (sc-20067, Cell Signaling); Caspase3 rAb (clone C92-605, RUO) (14C10, BD Biosciences); Caspase3 rAb (9662 S, Cell Signaling); active Caspase3 rAb (ab49822, Abcam); Desmoplakin rAb (sc-33555, Santa Cruz); Plakoglobin mAb (PG51, Progen); Dsc2 rAb (610120, Progen); Dsg2 mAb (33-3D) was kindly received from Prof. David Garrod; E-Cadherin mAb (HECD-1) (ab1416, Abcam); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rAb (14c10, Cell Signaling); HSC70 mAb (B6:sc-7298, Santa Cruz); β-actin mAb (8H10D10, Cell Signaling); anti-53BP1 (05–726, Upstate); anti-ATM (phospho S1981) (ab81292, Abcam); anti-ATM (ab32420, Abcam); anti-CHK2 (phospho T68) (ab32148, Abcam); anti-CHK2 (ab109413, Abcam).

Techniques: Immunohistochemistry, Staining, Positive Control

Journal: Cell Death & Disease

Article Title: The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53

doi: 10.1038/s41419-019-1988-0

Figure Lengend Snippet: a Confocal microscopy of NTERTs treated with the PV sera, dual labeled for Dsg3 and p53. Cells were seeded at confluent densities in KGM for overnight before being treated with PV sera (at 40% concentration in KGM) from a different cohort of PV patients ( n = 17), for 24 h. Disruption or depletion of Dsg3 at the plasma membrane accompanied with marked increases in p53 was observed in PV serum-treated cells that were abolished by p53 knockdown, compared to controls exposed to sera of healthy individuals that displayed, predominantly, nuclear p53 signals. Of note, p53 also showed distribution at the membrane where it colocalized with the fragmented Dsg3 (arrows in the inserts). Additional data for p53 knockdown was shown in Supplementary material Fig. S . The image magnification in PV serum-2 was doubled, relative to other panels, to highlight the disruption of junctions and peripheral distribution of p53. b Scatter and whisker plots of Dsg3 and p53 cellular and subcellular expression ( n = 16 for PV serum samples, n = 6 for control samples). Student t -test and the Wilcoxon–Mann–Whitney Rank Test were used for statistically significant analysis here and gave similar results. c The relative ratio of p53 nuclear versus cytoplasmic cellular distribution in controls and 16 PV sera treated NTERTs. d Treatment of NTERTs with the pathogenic monoclonal antibody AK23 targeting Dsg3 N-terminus, caused p53 induction, in a time and dose-dependent manner ( n = 11 fields per condition, pooled from 2 independent experiments). For the dose-response experiment, cells were treated with AK23 for 6 h. e Confocal images of cells with triple staining, treated in the presence and absence of AK23. Disruption of F-actin along with Dsg3 (arrowheads) was readily detectable in cells exposed to AK23. Increase p53 expression was detected predominantly in the nucleus and also was observed at the plasma membrane where it showed colocalization with Dsg3 and F-actin (arrows) in cells treated with AK23. The membrane distribution of p53 was not detectable in control cells. The protein colocalization of the dotted line box is shown at the bottom. (* p < 0.05, ** p < 0.01, *** p < 0.001). Scale bar, 10 µm. f Schematic model of Dsg3 in the suppression of p53 in keratinocyte response to stresses. Disruption of Dsg3 by RNAi or PV autoantibodies evokes p53 induction

Article Snippet: The following mouse (m) and rabbit (r) monoclonal/polyclonal antibodies (Abs) were used: Dsg3 mAb against the N-terminus (5H10) (sc-23912, Santa Cruz); Dsg3 rAb against the C- terminus (H145) (sc-20116, Santa Cruz); p53 mAb (DO-1) (ab1101, Abcam); p53 rAb (C-19) (sc-1311-R, Santa Cruz); MDM2 rAb (EP16627) (ab178938, Abcam); phospho MDM2 rAb (S166) (ab131355, Abcam); p21 WAF1/CIP1 rAb (C-19) (sc-397, Santa Cruz); Bax mAb (sc-20067, Cell Signaling); Caspase3 rAb (clone C92-605, RUO) (14C10, BD Biosciences); Caspase3 rAb (9662 S, Cell Signaling); active Caspase3 rAb (ab49822, Abcam); Desmoplakin rAb (sc-33555, Santa Cruz); Plakoglobin mAb (PG51, Progen); Dsc2 rAb (610120, Progen); Dsg2 mAb (33-3D) was kindly received from Prof. David Garrod; E-Cadherin mAb (HECD-1) (ab1416, Abcam); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rAb (14c10, Cell Signaling); HSC70 mAb (B6:sc-7298, Santa Cruz); β-actin mAb (8H10D10, Cell Signaling); anti-53BP1 (05–726, Upstate); anti-ATM (phospho S1981) (ab81292, Abcam); anti-ATM (ab32420, Abcam); anti-CHK2 (phospho T68) (ab32148, Abcam); anti-CHK2 (ab109413, Abcam).

Techniques: Confocal Microscopy, Labeling, Concentration Assay, Disruption, Clinical Proteomics, Membrane, Knockdown, Whisker Assay, Expressing, Control, MANN-WHITNEY, Staining

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: H460 cells were transiently transfected with GFP-siRNA-p120ctn plasmids or with empty vector as control. 24 hours after transfection, an aliquot of the cells was transfected again with p120ctn isoform 1A or 3A cDNA plasmids. ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal shown in the nucleus and cytoplasm indicates effective expression of GFP from GFP-siRNA-P120ctn, confirming the successful transfection. Depletion of p120ctn (I) reduced the E-cadherin levels (II). Transfection with empty vector (III) did not affect the E-cadherin levels (IV). Restitution of p120ctn isoform 1A (V) restored the E-cadherin on the cell membrane (VI), while restitution of p120ctn isoform 3A (VII) had no effects on E-cadherin levels (VIII). ( B ) Levels of E-cadherin were then analyzed by Western blot assay. The results confirmed that depletion of p120ctn resulted in decreased E-cadherin levels; restitution of p120ctn isoform 1A restored the E-cadherin levels, while restitution of p120ctn isoform 3A had no effects on E-cadherin expression. ( C ) The invasiveness of H460 cells were analyzed by Matrigel invasion assay. P120ctn ablation enhanced the invasiveness in comparison with the control group transfected with vector alone (*, P <0.01). Restitution of p120ctn isoform 1A reduced the invasiveness of H460 cells in comparison with the group with p120ctn ablation (Si-p120ctn) (**, P <0.01), while p120ctn isoform 3A increased the invasiveness (***, P <0.01).

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the membrane was incubated overnight at 4°C with either the mouse monoclonal antibody against p120ctn (1∶500, 610134, BD Transduction Laboratories, USA), E-cadherin (1∶200, SC-8426, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GFP (1∶400, SC-9996, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Membrane, Western Blot, Invasion Assay, Comparison

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: SPC cells were transiently transfected with GFP-siRNA-p120ctn or with empty vector as control. 24 hours after transfection, an aliquot of cells was transfected again with p120ctn isoform 1A or 3A cDNA plasmids. ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal shown in the nucleus and cytoplasm indicates effective expression of GFP from GFP-siRNA-P120ctn, confirming the successful transfection. Depletion of p120ctn (I) reduced the E-cadherin levels (II). Transfection with empty vector (III) did not affect the E-cadherin levels (IV). Restitution of p120ctn isoform 1A (V) restored the cytoplasmic E-cadherin levels (VI), while restitution of p120ctn isoform 3A (VII) had no effects on the E-cadherin levels (VIII). ( B ) Levels of E-cadherin were then analyzed by Western blot assay. The results confirmed that depletion of p120ctn resulted in decreased E-cadherin levels. Restitution of p120ctn isoform 1A restored the E-cadherin levels, while restitution of p120ctn isoform 3A had no effects on E-cadherin expression. ( C ) The invasiveness of SPC cells were analyzed by Matrigel invasion assay. P120ctn ablation reduced the cell invasiveness in comparison with the control group transfected with vector alone (*, P <0.01). Restitution of p120ctn isoform 1A and 3A both enhanced the invasiveness of SPC cells in comparison with the group with p120ctn ablation (Si-p120ctn) (**, P <0.01, ***, P <0.01).

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the membrane was incubated overnight at 4°C with either the mouse monoclonal antibody against p120ctn (1∶500, 610134, BD Transduction Laboratories, USA), E-cadherin (1∶200, SC-8426, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GFP (1∶400, SC-9996, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Western Blot, Invasion Assay, Comparison

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: H460 cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn, M1–M3, and M5-GFP was expressed in the nucleus and cytoplasm. GFP from GFP-si-P120ctn and M4-GFP was expressed in the nucleus, cytoplasm and on the cell membrane (IX). Expression or repletion of M4 mutant (IX) restored E-cadherin on the cell membrane (X), while expression of the other mutants had no significant effects on E-cadherin levels. ( B ) The levels of E-cadherin were then analyzed by Western blot assay. Expression of M1–M5 mutants were detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated the E-cadherin levels, whereas the other mutants had no effects on the E-cadherin levels. ( C ) The invasiveness of H460 cells was analyzed by Matrigel invasion assay. Repletion of M4 mutant reduced the cell invasiveness in comparison with the group of p120ctn ablation (Si-p120ctn) (**, P <0.01), while repletion of M2 mutant enhanced the invasiveness (*, P <0.01). Repletion of the other mutants (M1, M3 and M5) did not show significant effects on cell invasiveness in comparison with the group of 120ctn ablation.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the membrane was incubated overnight at 4°C with either the mouse monoclonal antibody against p120ctn (1∶500, 610134, BD Transduction Laboratories, USA), E-cadherin (1∶200, SC-8426, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GFP (1∶400, SC-9996, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Construct, Membrane, Mutagenesis, Western Blot, Invasion Assay, Comparison

Journal: PLoS ONE

Article Title: N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin

doi: 10.1371/journal.pone.0037008

Figure Lengend Snippet: SPC cells were transiently transfected with GFP-siRNA-p120ctn plasmids. 24 hours after transfection, an aliquot of the cells was transfected again with one of the five p120ctn isoform 1A deletion mutants M1–5 cDNA plasmids or with empty vector as control (The group transfected with vector alone was not included in the data). ( A ) Levels and localization of E-cadherin were analyzed by immunofluorescence. The green signal indicates expression of GFP from GFP-siRNA-P120ctn construct in image I and represents combined expression of GFP from GFP-siRNA-P120ctn and M1–5-GFP in images III, V, VII, IX and XI. GFP from GFP-si-P120ctn or M1–M5-GFP was expressed in the nucleus and cytoplasm. Expression (repletion) of M4 mutant (IX) restored cytoplasmic E-cadherin (X), while expression of the other mutants had no significant effects on the E-cadherin levels. ( B ) The levels of E-cadherin were analyzed by Western blot assay. Expression of M1–M5 mutants was detected by using antibody against GFP. The results showed that expression of M4 mutant up-regulated E-cadherin levels, whereas expression of the other mutants had no effects on the E-cadherin levels. ( C ) The invasiveness of SPC cells was analyzed by Matrigel invasion assay. Expression of M2 or M4 mutants enhanced the cell invasiveness in comparison with the group with ablated p120ctn (Si-p120ctn) (*, P <0.01, **, P <0.01), while expression of other three p120 1A mutants did not show significant effects.

Article Snippet: After transferring to a polyvinylidene fluoride (PVDF) membrane, the membrane was incubated overnight at 4°C with either the mouse monoclonal antibody against p120ctn (1∶500, 610134, BD Transduction Laboratories, USA), E-cadherin (1∶200, SC-8426, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GFP (1∶400, SC-9996, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Transfection, Plasmid Preparation, Control, Immunofluorescence, Expressing, Construct, Mutagenesis, Western Blot, Invasion Assay, Comparison